This proposal seeks to relate fundamental properties of membrane channels to atomic structure. The proposal builds on high level expression, functional assays, and a 2.2Angstrom structure of the aquaglyceroporin GIpF, as an archetype of the large aquaporin (AQP) family. An aim is to understand selectivity for glycerol, linear alditols and water using mutagenesis of side chains in the lumen of the channel. Assays of conductance, structure determination and molecular simulations are to be used throughout to test hypotheses and to refine most critical experimental tests. Mechanisms by which AQPs absolutely exclude proton conductance are to be defined. The mechanism of pH induced channel gating in AQP3 is to be mimicked by mutational grafting into GIpF, and described in atomic and electrostatic terms. The ion channel present in AQP6 is to be defined by mutagenesis and implanted into GIpF. Determinants of ion channel selectivity, primarily encoded by side chains in GIpF, and regulation by pH will be probed by mutagenesis, ion channel conductance, and three dimensional structure analysis at atomic resolution. Mutations in clinically important AQPs are to elucidate mechanism, and to serve as a template for drug design. Mutations in AQP2 that are the source of diabetes insipidus are to be grafted into GIpF to define their effects on channel properties versus trafficking. The phosphoregulation mechanism of AQP2 is to be grafted into GIpF and defined mechanistically. Other AQPs of clinical relevance, one from a human pathogen and AQP0 from eye lens are to be crystallized for structure determination. Four species of the ammonia channel family are to be expressed to initiate crystallization of a new family of channels. A further aim is to improve crystals of the Acetylcholine receptor as a means of three-dimensional structure determination of an archetype of gating mechanisms in an archetype of neuroreceptor superfamilies.